The present invention provides recombinant DNA compounds and host cells containing novel polyketide synthase (PKS) genes and novel polyketides. The invention relates to the fields of chemistry, medicinal chemistry, human and veterinary medicine, molecular biology, pharmacology, agriculture, and animal husbandry.
Polyketides are structurally diverse natural products that include important therapeutic agents used as antibacterials (erythromycin), inmmunosuppressants (FK506), cholesterol-lowering agents (lovastatin), and others (see Katz et al., 1993, Polyketide synthesis: prospects for hybrid antibiotics, Annu. Rev. Microbiol. 47: 875-912, incorporated herein by reference). Currently, there are about 7,000 identified polyketides, but this represents only a small fraction of what nature is capable of producing.
DNA sequencing of genes encoding several of the enzymes that produce type 1 modular polyketide synthases (PKSs) has revealed the remarkably logical organization of these multifunctional enzymes (see Cortes et al., 1990, An unusually large multifunctional polypeptide in the erythromycin-producing polyketide synthase of Sacciaropolyspora erythraea, Nature 348: 176-178; Donadio et al., 1991, Modular organization of genes required for complex polyketide biosynthesis, Science 252: 675-679; Schwecke et al., 1995, The biosynthetic gene cluster for the polyketide immunosuppressant rapamycin, Proc. Natl. Acad. Sci. USA 92: 7839-7843; and August et al., 1998, Biosynthesis of the ansamycin antibiotic rifamycin: deductions from the molecular analysis of the rif biosynthetic gene cluster of Amycolatopsis mediterranei S699, Chem Biol 5: 69-79, each of which is incorporated herein by reference). The application of innovative combinatorial techniques to this genetic organization has prompted the generation of novel natural products, by adding, deleting, or exchanging domains or entire modules. See U.S. Pat. Nos. 5,672,491; 5,712,146; 5,830,750; 5,843,718; 5,962,290; and 6,022,731, each of which is incorporated herein by reference. It would be advantageous to have a practical combinatorial biosynthesis technology that could achieve and perhaps exceed the diversity of modular polyketide structures thus far revealed in nature.
The known modular PKSs have a linear organization of modules, each of which contains the activities needed for one cycle of polyketide chain elongation, as illustrated for 6-deoxyerythronolide B synthase (DEBS) in FIG. 1A. The minimal module contains a ketosynthase (KS), an acyltransferase (AT), and an acyl carrier protein (ACP) that together catalyze a 2-carbon extension of the chain. The specificity of the AT for either malonyl or an alpha-alkyl malonyl CoA determines which 2-carbon extender is used, and thus the nature of the alkyl substituent at the alpha-carbon of the growing polyketide chain. After each 2-carbon unit condensation, the oxidation state of the beta-carbon is either retained as a ketone, or modified to a hydroxyl, methenyl, or methylene group by the presence a ketoreductase (KR), a KR+ a dehydratase (DH), or a KR+DH+ an enoyl reductase (ER), respectively. In effect, the AT specificity and the composition of catalytic domains within a module serve as a xe2x80x9ccodexe2x80x9d for the structure of each 2-carbon unit. The order of the modules in a PKS specifies the sequence of the distinct 2-carbon units, and the number of modules determines the size of the polyketide chain.
The remarkable structural diversity of polyketides (see O""Hagan, The Polykehde Metabolites; Ellis Horwood, Chichester, 1991, incorporated herein by reference) is governed by the combinatorial possibilities of arranging modules containing the various catalytic domains, the sequence and number of modules, and the post-PKS xe2x80x9ctailoring enzymesxe2x80x9d that accompany the PKS genes. The direct correspondence between the catalytic domains of modules in a PKS and the structure of the resulting biosynthetic product allows rational modification of polyketide structure by genetic engineering.
Over the past several years, examples of modifying each of the elements that code for polyketide structure has been accomplished (see Kao et al., 1996, Evidence for two catalytically independent clusters of active sites in a functional modular polyketide synthase, Biochemistry 35: 12363-12368; Liu et al., 1997, Biosynthesis of 2-nor-6-deoxyerythronolide B by rationally designed domain substitution, J. Am. Chem. Soc. 119: 10553-10554; McDaniel et al., 1997, Gain-of-function mutagenesis of a modular polyketide synthase, J. Am. Chem. Soc. 119: 4309-4310; Marsden et al., 1998, Engineering broader specificity into an antibiotic-producing polyketide synthase, Science 279: 199-202; and Jacobsen et al., 1997, Precursor-directed biosynthesis of erythromycin analogs by an engineered polyketide synthase, Science 277: 367-369, each of which is incorporated herein by reference).
Recently, a combinatorial library of over 50 novel polyketides was prepared by systematic modification of DEBS, the PKS that produces the macrolide aglycone precursor of erythromycin (see U.S. patent application Ser. No. 09/429,349, filed Oct. 28, 1999; PCT patent application US99/24483, filed Oct. 20, 1999; and McDaniel et al., 1999, Multiple genetic modification of the erythromycin gene cluster to produce a library of novel xe2x80x9cunnaturalxe2x80x9d natural products, Proc. Natl. Acad. Sci. USA 96: 1846-1851, each of which is incorporated herein by reference). With a single plasmid containing the eryAI, -AII and -AIII genes encoding the three DEBS subunits, ATs and beta-carbon processing domains were substituted by counterparts from the rapamycin PKS (see Schwecke et al., 1995, supra) that encode alternative substrate specificities and beta-carbon processing activities. The approach used was to develop single xe2x80x9cmutationsxe2x80x9d, then sequentially combine the single mutations to produce multiple changes in the PKS. It was observed that when two or more single PKS mutants were functional, there was a high likelihood that combinations would also produce the expected polyketide. Although this strategy provided high assurance that the multiple mutants would be productive, the production of each polyketide required a separate engineering. Thus, if X mutants of eryAI, Y mutants of eryAII, and Z mutants at eryAIII were prepared, X+Y+Z separate experiments were required to produce that same number of polyketides. Clearly, the preparation of very large libraries by this approach is laborious.
Another strategy for preparing large numbers of polyketides is by random digestion-religation leading to xe2x80x9cmutagenesisxe2x80x9d of the domains or modules of a mixture of PKS genes, including the refinements embodied in the DNA shuffling method (see Patten et al., 1997, Applications of DNA shuffling to pharmaceuticals and vaccines, Curr. Op. Biotechnol. 8: 724-733, incorporated herein by reference). The expected low probability of assembling an active PKS by such an approach, however, would demand an extraordinary analytical effort (in the absence of a biological selection) to detect clones that produced polyketides within the much larger number of clones that are non-producers.
There remains a need for practical approaches to create large libraries of polyketides, non-ribosomal peptides, and mixed polyketides/non-ribosomal peptides.
The present invention provides a method for expressing a polyketide or non-ribosomal peptide in a host cell employing a multiplicity of recombinant vectors, which may be integrative or freely replicating. Each of the multiplicity of vectors encodes a portion of the polyketide synthase or non-ribosomal peptide synthase that produces the polyketide or non-ribosomal peptide. In one embodiment, at least one of the multiplicity of vectors encodes one or more proteins that further modify the polyketide or non-ribosomal peptide produced.
In a preferred embodiment, the vectors replicate in and/or integrate into the chromosome of a Streptomyces host cell. Preferred integrating vectors include vectors derived from pSET152 and pSAM2. Preferred replicating vectors include those containing a replicon derived from SCP2* or pJV1.
In another embodiment, the present invention provides novel polyketides. Such novel polyketides include those shown in FIG. 3 as compound nos. 29-43 and 45-59. Other novel polyketides of the invention include the polyketides obtainable by hydroxylation and/or glycosylation of compounds 29-43 and 45-59. Preferred compounds of the invention include those 14-membered macrolactones with a C-6 and/or C-12 hydroxyl and/or a C-3 and/or C-5 glycosyl, including but not limited to those with a desosaminyl residue at C-5 and a cladinosyl residue at C-3.
These and other embodiments, modes, and aspects of the invention are described in more detail in the following description, the examples, and claims set forth below.